Campylobacter spp. isolated from poultry in Iran: Antibiotic resistance profiles, virulence genes, and molecular mechanisms

Abstract Campylobacter spp. genera is one of the most common causes of microbial enteritis worldwide. The objective of this work was to investigate the antimicrobial resistance (AMR) patterns, virulence genes, and genetic variation of thermophilic Campylobacter species collected from chicken meat samples in Iran. A total of 255 meat specimens were taken and transferred to the laboratory. Culture methods were utilized to identify the Campylobacter genus, and PCR and sequencing were performed to confirm the organisms. Antimicrobial susceptibility evaluation was performed using broth microdilution for six antimicrobials [ciprofloxacin (CIP), nalidixic acid (NAL), sitafloxacin (SIT), erythromycin (ERY), tetracycline (TET), and gentamicin (GEN)]. By using PCR, AMR and virulence genes were detected. The detection rate of Campylobacter spp. was 64 (25.09%) out of 255 meat samples, with C. jejuni and C. coli accounting for 41 (64.06%) and 14 (21.87%), respectively. Other Campylobacter isolates accounted for 14.06% of the total (nine samples). The antibiotic susceptibility of all Campylobacter isolates was tested using six antibiotics, and all (100%) were resistant to CIP and NAL. However, TET resistance was observed in 93.9% and 83.3% of C. jejuni and C. coli isolates, respectively. Four (8.2%) C. jejuni isolates were multidrug‐resistant (MDR), while none of the C. coli isolates were MDR. Two of the four MDR isolates were resistant to CIP, NAL, TET, and ERY, whereas the other two isolates were resistant to CIP, NAL, TET, and GEN. The values of the Minimum Inhibitory Concentration (MIC) were as follows: CIP, 64–256 μg/ml; NAL, 128–512 μg/ml; TET, 2–1024 μg/ml; SIT, 0.25–1 μg/ml; ERY, 1–32 μg/ml; and GEN, 1–256 μg/ml. recR, dnaJ, cdtC, cdtB, cdtA, flaA, ciaB, cadF, and pidA were discovered in more than 50% of C. jejuni isolates, although wlaN, virbll, cgtB, and ceuE were found in <50%. flaA, cadF, pidA, and ciaB were discovered in more than 50% of the C. coli samples, whereas recR, cdtC, cdtB, cdtA, and cgtB were found in less than half. For C. coli, the percentages for wlaN, dnaJ, virbll, and ceuE were all zero. The results of this study show Campylobacter isolates obtained from poultry have higher resistance to quinolones and TET, pathogenicity potential, and varied genotypes.


| INTRODUC TI ON
Campylobacter jejuni and Campylobacter coli are foodborne pathogens that cause gastroenteritis (campylobacteriosis) in humans, affecting an estimated 95 million people each year throughout the world (Facciolà et al., 2017;Jonaidi-Jafari et al., 2016;Torkan et al., 2018). Campylobacter infection is far more prevalent in affluent nations than foodborne diseases caused by Listeria monocytogenes, Escherichia coli O157, Vibrio cholerae O139, and Shigella, with an estimated 1.3 million foodborne cases annually in the United States alone (Hansson et al., 2018;Raissy et al., 2014;Rahimi et al., 2017). C. jejuni and C. coli are the major species causing sporadic gastroenteritis throughout the world Ommi et al., 2017). C. jejuni is responsible for 90% of campylobacteriosis cases (Facciolà et al., 2017;Kaakoush et al., 2015). C. coli comes second and accounts for 5%-10% of cases (Tam et al., 2003). In 2018, 58% of C. jejuni outbreaks were linked to chicken and poultry products, according to reports (Tedersoo et al., 2022). The consumption of tainted poultry products is frequently associated with outbreaks.
Poultry is considered a reservoir host of C. jejuni because this pathogen is found mostly in the gastrointestinal system of chickens and has been linked to meat contamination during processing de Melo et al., 2021). Poultry products are a popular source of proteins, vital amino acids, mineral salts, and vitamins for many individuals (Alagawany et al., 2021). The primary sources of human campylobacter infections are raw meat handling and uncooked meat-eating. In controlled slaughtering procedures, chickens are slaughtered, skinned, and cut to bits by hand (Alagawany et al., 2021;Beiranvand et al., 2022;Cardoso et al., 2021). During the evisceration procedure, the corpse is drained, the visceral contents are separated, and the liver, heart, and intestines are gathered (Corry & Atabay, 2001). The discharge of digestive contents has the potential to infect these tissues. Since the demand for poultry products has increased in the world, ensuring their health and quality is the most important issue (Wickramasuriya et al., 2022). Food fraud, feces, microbiological contamination, physical faults, and product quality must all be examined regularly in poultry goods (Visciano & Schirone, 2021). A low C. jejuni prevalence (a percentage/proportion of colonized chickens in a flock) between chicken flocks or a 1 to 2 log10 reduction of C. jejuni loads in broiler intestines could lead to a decrease in public health risk, according to previous study's quantitative risk assessment and regression models (Pumtang-On et al., 2021). As a result, lowering C. jejuni levels and preventing campylobacter colonization of chickens on farms are the most effective ways to limit the risk of campylobacter contamination of chicken meat (Hakeem & Lu, 2021). Biosecurity monitoring, feed additives, drinking water sanitation, and the use of bacteriophages, probiotics, and bacteriocins are among the strategies that researchers have attempted to develop and assess in primary broiler production so far Shehata et al., 2022). Even though several of these interventions have resulted in large decreases in C. jejuni burdens in chicken intestines, none of them have completely eradicated or prevented C. jejuni colonization in poultry (Hermans et al., 2011;Rahimi et al., 2014).
Campylobacter jejuni survival and virulence are mediated by motility, adhesion, quorum sensing, and biofilm formation. Although the importance of C. coli may seem small in percentage terms, the increasing rate of antibiotic resistance observed and the difference in risk factors for infection between the two species means that it is usually studied in the same way as C. jejuni .
While most Campylobacter infections induce mild diarrhea, severe, chronic, or systemic infections (such as bacteremia) can occur in young children, the elderly, and individuals with immunodeficiency disorders (Banisharif et al., 2019;Zarinnezhad et al., 2021). Under these circumstances, antibiotic therapy is required which may need the prescription of fluoroquinolone (FQ), macrolide, or aminoglycoside antibiotics (Dai et al., 2020). But Campylobacter has acquired multiple resistance mechanisms in response to antibiotic use in clinical settings and animal husbandry, and antibiotic-resistant Campylobacter is becoming more common, compromising the efficacy of antibiotic therapy and creating a severe public health risk (Rossi et al., 2021). In Iran, there have been few studies on the prevalence and antibacterial resistance of intestinal Campylobacteriosis in people and poultry products. The lack of a broad monitoring program limited the availability of regular Campylobacter spp. culture supplies. Therefore, the present study aims to investigate the antimicrobial resistance (AMR) patterns, virulence genes, and genetic variation of thermophilic Campylobacter species collected from chicken meat samples in Iran.

| Extraction of DNA and PCR identification of genus
The Qiagen QIAamp PowerFecal Kit (Qiagen) was used to extract genomic DNA from pure cultures, as directed by the manufacturer.
Then, utilizing genus-specific primers (C412F and C1228R), C. jejuni cj0414 gene primers (C1 and C3), and C. coli ask gene primers (CC18F and CC519R), a multiplex PCR was carried out (Yamazaki-Matsune et al., 2007). The primers were chosen for their ability to distinguish between Campylobacter genus and species. 12.5 μl 2X Master Mix (Thermo Fisher Scientific), 1 μl primer (10 μM), 1.5 μl template DNA (20 μg/ml), and 7 μl sterile deionized water were used to make the PCR mixture (25 μl). The MiniAmp Plus Thermal Cycler was used to do one cycle of 95°C for 5 min, 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s, and a final extension of 72°C for 5 min (Applied Biosystems). Before being analyzed, the PCR products were kept at 4°C.

| Detection of AMR genes
Genes-encoding AMR was determined by PCR experiments using primer in Table 1. The investigated genes were: aphA-3-1 (GEN resistance), cmeB (efflux pump), blaOXA-61 (ampicillin (Beta-lactam) resistance), tet(O) (TET resistance), and aadE1 (aminoglycosides resistance; Table 1). After electrophoresis, bands of PCR products were observed under ultraviolet light using a Dual UV Transilluminator (Core BioSystem). The above-mentioned approach was used to carry out the PCR procedure. After electrophoresis, bands of PCR products were visible under ultraviolet light using a Dual UV Transilluminator (Core BioSystem). The amplification products' bands were evaluated by comparing them to a 100-bp DNA ladder (Dyne bio; Table 2). The AMPure XP beads (Beckman Coulter) were used to purify antibioticresistant gene PCR products, which were then sequenced using the Sanger technique at SolGent (Solutions for Genetic Technologies).

| Statistical analysis
All data were input into a Microsoft Excel Sheet (Microsoft Corp.) and analyzed with SPSS version 20 data analysis. The connections were evaluated using the Chi-square test and logistic regression. A p-value of <.05 was statistically significant for all experiments.

| Susceptibility testing for antimicrobials
The antibiotic susceptibility of all Campylobacter isolates was tested using six antibiotics, and they all exhibited significant levels of resistance to CIP, NAL, and TET. Ciprofloxacin and NAL resistance were TA B L E 1 Primer sequences for the multiplex PCR experiment Results showed that most of the C. coli isolates obtained from meat samples were resistant to at least three antibiotics. In fact, these isolates showed the MDR phenotype. There was a statistical difference between the specimens and AMR incidence (p < .05).

Detection of antibiotic-resistant genes showed that tet(O), bla-
OXA-61 were present in all isolates. However, there were no bands for the multidrug efflux pump gene (cmeB) or the aphA-3-1 gene in any of the strains. blaOXA-61 and aadE1 genes were found to be most common in C. coli isolates (  recR, dnaJ, cdtC, cdtB, cdtA, flaA, ciaB, cadF, and pidA were discovered in more than 50% of C. jejuni isolates, although wlaN, virbll, cgtB, and ceuE were found in <50% (Table 6). flaA, cadF, pidA, and ciaB were discovered in more than 50% of the C. coli samples, whereas recR, cdtC, cdtB, cdtA, and cgtB were found in less than half. For C. coli, the percentages for wlaN, dnaJ, virbll, and ceuE were all zero ( Table 7).

| DISCUSS ION
Poultry contamination with Campylobacter at the farm level typically impacts the entire commercial poultry system from farm to fork, necessitating appropriate treatments to prevent transmission from poultry to people Wang et al., 2016).
Campylobacter was found in 25.09% of cases, with C. jejuni (64.06%) outnumbering C. coli (21.87%). The diagnostic accuracy was somewhat greater than that reported earlier for layers in the United States (Novoa Rama et al., 2018) but lower than that previously reported for layers in the Netherlands (Schets et al., 2017) and Sri Lanka (Kalupahana et al., 2013). C. jejuni is the most common cause of human campylobacteriosis, according to the literature (Wei et al., 2014), and our findings back up this claim. Furthermore, there have been instances where C. coli was the dominant or only species identified (Silva et al., 2011;Vickers, 2017;Wei et al., 2014).
Several Campylobacter species showed significant levels of resistance to antimicrobial drugs (CIP, NAL, and TET), which is consistent with prior observations in Korea (Lee et al., 2017) and elsewhere (Elhadidy et al., 2018;Zhang et al., 2020). The use of CIP was outlawed in Korea in 2010 (Ku et al., 2011), although mass injection of chickens with FQs, particularly enrofloxacin, is still permitted (Jiang et al., 2008). Despite the lack of quinolone usage, quinoloneresistant Campylobacter bacteria have been detected in Australia (Abraham et al., 2020). Although the fact that FQs are outlawed in numerous countries, resistant strains persist in bacterial communities, which might explain why they continue to be found in people and animals (Sproston et al., 2018). Considering the poultry manufacturing systems, monitoring methods, and geographical region, the link between the use of FQs in livestock farming and the increasing prevalence of AMR infections varies (Fan et al., 2018). The rising incidence of human campylobacteriosis in Asia, Europe, and America is thought to be fuelled in part by a worldwide presence of C. jejuni isolates resistant to quinolones transmitted through poultry . Strong resistance to TET was found, which is consistent with similar accounts from Korea (Lee et al., 2017), China , and India (Kabir et al., 2015). Because of its low cost and ease of administration to animals through drinking water, TET is commonly used in the poultry and pig sectors (Jonker & Picard, 2010). SIT (MIC values: 0.125-1 mg/ml) was shown to be effective against all strains. SIT has unique properties not seen in other quinolones, such as a cyclopropyl circle with fluorine at R-1 and a chloride substituent at R-8, which may describe its remarkable potency (Changkwanyeun et al., 2016). Previous publications with a MIC of 0.25 mg/ml (Özmerdiven et al., 2015;Yabe et al., 2010) corroborate our findings. SIT is a contender for campylobacteriosis clinical testing (Özmerdiven et al., 2015), and is used to eradicate MDR Helicobacter pylori (Pohl et al., 2019). This might be a game-changer, considering there are few other treatments for severe campylobacteriosis (Pavlova et al., 2020).
The strains in this investigation showed minimal resistance to ERY and GEN, which has been documented in other Asian countries such as Vietnam (Carrique-Mas et al., 2014), and China (41). In China , the United States (Tang et al., 2017), and Europe, resistance to ERY has been modest and consistent. The limited tolerance to ERY might be partially explained by delayed development of resistant isolates when treated for ERY, as well as decreased resistance variant survivability (Luangtongkum et al., 2012). Because GEN is used to treat systemic infections, resistance to it has been limited. The use of current antimicrobial drugs with caution, as well as initiatives to develop novel alternative treatment alternatives, might assist to slow the spread of AMR (Lynch et al., 2020).
The virulence of Campylobacter species depends on their virulome (Han et al., 2019). recR, dnaJ, cdtC, cdtB, cdtA, flaA, ciaB, cadF, and pidA were detected in more than 50% of C. jejuni Type of meat C. coli aphA-3-1 cmeB tet(O) blaOXA-61 aadE1 were found in <50%. The diagnostic accuracy was comparable to those reported in a recent report from Korea , but greater than those in South Africa (Otigbu et al., 2018;Pillay et al., 2020) and Chile (González-Hein et al., 2013). Any strain of bacteria appears to require cadF in order to invade epithelial TA B L E 6 Distribution of genotypes amongst the Campylobacter jejuni strains isolated from different types of raw meat samples  (Ramires et al., 2020). The cellular membranes phospholipase A (pldA) was found more in C. jejuni than in C. coli, corroborating South African research (Pillay et al., 2020), whereas our findings for ciaB are higher than those of a prior Korean investigation . In cellular models, the expression of cadF and ciaB aids Campylobacter adherence and internalization (Ramires et al., 2020). C. jejuni had a somewhat greater csrA detection accuracy than C. coli, although csrA was absent in Campylobacter isolates from South Africa (Otigbu et al., 2018). ggt was not found in any of our samples. Although the latter was reported to be just 5.5% in Chile (Otigbu et al., 2018), our findings contrast with Finland's high values (30.9%-43.2%; Gonzalez et al., 2009). The disparity might be explained by the intricacy of the colonization process, which involves many genes and the utilization of isolates from a single sampling location (González-Hein et al., 2013).
Moreover, MDR and virulent C. jejuni isolates were detected more frequently in the summer than in the winter, demonstrating that temperature has a role in the expression of certain genes (Kim et al., 2019). We also mention that numerous virulence genes are plasmid-mediated, which might have an impact on their presence in various isolates. The virulence genes identified in this work had previously been found in Campylobacter strains isolated from people (Abraham et al., 2020;Kim et al., 2019), indicating that these Campylobacter isolates may be virulent enough to cause human illnesses.
In this study, Campylobacter isolates were characterized by the detection of certain resistance and virulence factors, which is limited to understanding the mechanisms of resistance and virulence.
The results of whole genome sequencing analysis can determine the epidemiology and evolutionary pathways of Campylobacter spp to better tailor interventions to reduce cases of campylobacteriosis in Iran.

| CON CLUS ION
In conclusion, Campylobacter spp. collected from hens in this study showed significant antibiotic resistance and harbored various virulence and AMR genes. These strains could pose a risk to public health. The intensive use of antibiotics in chicken farming is to blame for the increase in MDR Campylobacter isolates. Antibiotic resistance in pathogenic bacteria can be reduced by monitoring antibiotic resistance in Campylobacter and by using antimicrobials appropriately in feed production.

ACK N OWLED G EM ENTS
The authors would like to thank Dr. Reza Sherafati for his assistance in sample collection. This work was financially supported by the Islamic Azad University, Shahrekord Branch, Shahrekord, Iran.

FU N D I N G I N FO R M ATI O N
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data analyzed during this study are included in this published article.

E TH I C A L A PPROVA L
The research was extracted from the Ph.D thesis in the field of Food Hygiene, and was ethically approved by the Council of